Delivery vehicle for probiotic bacteria comprising a dry matrix of polysaccharides, saccharides and polyols in a glass form and methods of making same

ABSTRACT

The disclosure relates to a solid glass matrix of polysaccharides, monossaccharides or disaccharides in combination with polyols as delivery vehicles for preservation and post gastric administration of a probiotic. The delivery vehicle is capable of releasing the probiotic at their site of action. The present invention further includes methods of making and using the solid glass matrix delivery vehicle of the invention.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation-in-part application of andclaims priority to copending application U.S. patent application Ser.No. 13/351,343 filed on Jan. 17, 2012 which is a divisional applicationof and claims priority to U.S. patent application Ser. No. 12/159,407filed on Nov. 21, 2008, now U.S. Pat. No. 8,097,245, which in turnclaimed priority to PCT Application No. PCT/US2006/049434 filed in theU.S. Patent and Trademark Office, PCT Division, on Dec. 28, 2006, whichin turn claimed priority to U.S. Provisional Patent Application No.60/754,502 filed on Dec. 28, 2005, the contents of all applications arehereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

The disclosure relates generally to the field of a delivery vehicle forprobiotic bacteria comprising a dry matrix of polysaccharides,saccharides and polyols in a glass form. Methods of making and usesthereof are also provided.

Probiotics are defined as live microbes that beneficially affect thehost by modulating mucosal and systemic immunity, as well as improvingintestinal function and microbial balance in the intestinal tract.Various nutritional and therapeutic effects have been ascribed toprobiotics including: modulating immune response, lowering serumcholesterol concentrations, improving lactose intolerance symptoms,increasing resistance to infectious intestinal diseases, decreasingduration of diarrhea, reducing blood pressure, and helping to preventcolon cancer (Isolauri E et al. 2001, Kailasapathy K and J. 2000,Marteau P R et al. 2001, Perdigon G et al. 2001). In order to exerttheir beneficial effects on the host, probiotics must remain viable andreach the intestine in large numbers (Favaro-Trindade and Grosso 2002).However, maintaining long term stability of probiotics requires specialstorage conditions, since viability deteriorates rapidly over a shorttime period at ambient temperature and humid conditions (Shah 2000). Inaddition to poor shelf life, a significant loss of viability occurs uponexposure of the probiotics to gastric conditions of low pH and digestiveenzymes. Existing preservation methods fail to provide satisfactoryviability upon storage and gastric protection, especially if cells arestored at ambient or higher temperature and humidity.

Freeze-drying is often used for preservation and storage of bacteriabecause of the low temperature exposure during drying. However, it hasthe undesirable characteristics of significantly reducing viability aswell as being time and energy-intensive. Freeze-drying involves placingthe cells in solution, freezing the solution, and exposing the frozensolid to a vacuum under conditions wherein it remains solid and thewater and any other volatile components are removed by sublimation.Standard freeze drying temperature of −30° C. to −70° C. are below thefreezing point of water, but are well above the glass transition (Tg)temperature of the drying solution, which results in the undesirableeffect of crystallization of water into ice. Freezing bacterial culturesresults in substantial physical damage to the bacterial cell wall andsubsequent loss of viability. Therefore, avoiding ice formation duringcold storage of proteins, viruses, cells, tissues, and organs is animportant problem in cryobiology.

The freezing point of water can be lowered by adding solutes that lowerthe vapor pressure of water. Freezing point depression is the physicalbasis on which essentially all currently used antifreeze agents (e.g.,glycols, sugars and salts) perform. The disadvantage of freezing pointdepressors, known as cryoprotectants, is that large quantities ofsolutes (10% or more) are required to lower the freezing point by even afew degrees Celsius. At sufficiently high concentrations (typically 50%or more), conventional antifreeze agents can prevent ice formation,allowing aqueous solutions to be cooled to temperatures well below 0° C.without freezing. However, cryoprotectants are generally toxic at thehigh concentrations required to achieve glass formation orvitrification.

Other methods used to prepare dry and stable preparations of probioticssuch as desiccation at ambient temperature and spray drying also hasdrawbacks. Desiccation at low or ambient temperature is slow, requiresextra precautions to avoid contamination, and often yieldsunsatisfactory viability. Spray drying involves short excursions torelatively high processing temperatures and results in viability lossesand limited storage times, even when stabilizing excipients are used(Lievense L C, van 't Riet K. 1994).

A viable and stable formulation for intestinal targeting of probioticshas been described by Simmonds et al. (2005). The process requires thegranulation of lyophilized bacteria with microcrystalline cellulosestabilizers such as skim milk, salts or short chain sugars and adisintegrant such as starch or alginic acid. The granulated semi drybacteria are then desiccated at 40-70° C. to reduce the residualmoisture level to less than 2 percent. This is followed by coating withan enteric agent and plasticizer. This multi-step process results inlarge particle size (over 425 micron) and still results in up to 1.5logs loss of viability. An additional disadvantage of this method is thehigh content of the enteric coating agents (over 25% of the microsphereweight), which are mostly synthetic and not recognized as food gradematerials. An inherent disadvantage of a coating procedure is that therelative proportion of the coating to active agent goes up by a cubicfunction of the particle, as the particle size gets smaller, making theprocess less usable for the production of particles of sizes less than300 micron.

An alternative method of bacterial preservation has been described whichuses a foam formation technique while eliminating the formation of icecrystals (Bronshtein et al. 2004, Roser et al. 2004). This methodrequires high concentrations of sugars (a combination of methylatedmono, di- and oligo-saccharides) in the drying media and a freeze drierthat is equipped with a controlled vacuum system and temperatureexposure, and the addition of foam forming elements and stabilizers. Inspite of some advantages of this method in achieving longer shelf lifestability, the foam-preserved bacteria are not protected from gastricexcursion. Furthermore this process is difficult and costly to scale upbecause the foam requires, by definition, large volumes of space underreduced atmospheric pressure (i.e., in a vacuum) for the production ofvery little mass. In addition, this material is very sensitive tohumidity and the product will take up water readily, decreasing theviability of the bacteria.

A composition containing a sugar (trehalose) partly in amorphous glassyphase and partly in crystalline hydrate phase has been proposed byFranks et al (2003). The crystalline hydrate phase serves as an agent todehydrate the amorphous phase, thereby enhancing the glass transitiontemperature of the amorphous glassy state. This composition was shown tostabilize single molecules such as proteins or nucleotides. The glasstransition temperature of a mixture depends, among other factors, on itschemical composition (sugars, proteins, salts) and the moisture content,with water acting as a plasticizer, depressing the glass temperature.If, at any time, the glass transition temperature (Tg) is exceeded,either by exposure to heat or in consequence of moisture migration intothe product, the amorphous glassy state may become liable toirreversible phase separation by crystallization. If crystallizationoccurs, any residual amorphous phase will then be composed of the othercomponents and the moisture, resulting in a further depression of theglass transition temperature.

A glass is an amorphous solid state that is obtained by controlleddesiccation of a solution. The advantage of the glassy phase inachieving long term stability results from the fact that diffusion inglassy (vitrified) materials occurs at extremely low rates (e.g.,microns/year). Glassy materials normally appear as homogeneous,transparent, brittle solids, which can be ground or milled into apowder. The optimal benefits of vitrification for long-term storage areobserved under conditions where Tg is greater than the storagetemperature. The Tg is directly dependent on water activity andtemperature, and may be modified by selecting an appropriate combinationof solutes (i.e., polysaccharides, sugars, salts and proteins).

Glass formation occurs naturally in some plant and arthropod speciesthat are very desiccation tolerant. A number of mosses and ferns,so-called resurrection plants, can undergo severe desiccation andsurvive for many years in a quiescent metabolic state only to reviveupon the return of water to the environment. In most cases, theadaptation characteristic is to increase internal concentrations ofcertain saccharides such as trehalose to a level that form glassystates.

Prior to the current disclosure, no one has been able to provide acommon and cost effective solution to the separate problems facing theprobiotic industry, namely maintaining long shelf life stability {i.e.,viability) of bacterial cells at ambient temperatures and high wateractivities (or high humidity) and providing gastric protection tominimize losses of probiotic viability during the transit through thestomach. The present invention overcomes these problems.

BRIEF SUMMARY OF THE INVENTION

The present invention encompasses compositions and methods of producingmicroparticles comprising a solid matrix in a glass form suitable fororal delivery. The compositions include a combination of apolysaccharide, a saccharide, a polyol and a probiotic bacteria. Thesecompositions are designed to provide longer shelf life stability atambient temperature in high water activity environments, and gastricprotection of the probiotic. Furthermore, the method of production ofthis matrix involves processes that result in a minimal loss ofprobiotic viability.

Accordingly, one aspect of the invention comprises a preservationmixture of carbohydrates including at least one polysaccharide, onesaccharide (di- or oligo saccharide) and one polyol and at least onebacterium to be incorporated in the carbohydrate mixture.

In a preferred aspect, the bacteria in the preservation carbohydratemixture are probiotic bacteria selected from, but not limited to thegroup consisting live Lactobacillus, Bifidobacterium, Enterococcus,Propionobacterium, Bacillus, and Streptococcus.

In another aspect of the invention the polysaccharide in thepreservation mixture provides gastric protection and control releasemechanism that gradually release the microbes at their site of actionalong the fore and hind gut of the animal or man. Examples ofpolysaccharides with gastric protection and a controlled releasemechanism are hydrocolloid forming polysaccharides selected from thegroup including, but not limited to starch (including non-digestiblestarch), pectin, inulin, xanthan gum, alginate, alginic acid, chitosan,carrageenan, carboxymethyl cellulose, methyl cellulose, guar gum, gumarabic, locust bean gum and combinations thereof. Also preferably, theconcentration of the polysaccharides in the preservation mixture is lessthan 10% w/v and more preferably less than 5% w/v of the preservationmixture.

In another aspect of the invention the saccharide/polyol combination inthe preservation mixture is formulated so that it does not crystallizeduring drying and long-term storage at ambient temperature. A suitableglass formulation system includes, but is not limited to,trehalose/glycerol, trehalose/mannitol, trehalose/maltitol,trehalose/isomalt, trehalose/adonitol, trehalose/lactitol,trehalose/sorbitol, sucrose/glycerol, sucrose/mannitol,sucrose/maltitol, sucrose/isomalt, sucrose/adonitol, sucrose/lactitol,sucrose/sorbitol, inulin/glycerol, inulin/mannitol, inulin/maltitol,inulin/isomalt, inulin/adonitol, inulin/lactitol, and inulin/sorbitol.Trehalose is a naturally occurring, non-reducing disaccharide, which isassociated with the prevention of desiccation damage in certain plants,microbes and animals that can dry out without damage and revive whenrehydrated. Trehalose also has been shown to be useful in preventingdenaturation of proteins, viruses and foodstuffs during desiccation(Chen et al. 2001, Crowe and Crowe 1992, Liao et al. 2002). Compared tosucrose, the glass transition temperature of trehalose is significantlyhigher (110° C. vs only 65° C.) (Crowe et al. 1998). However, asaccharide alone is not always sufficient to stabilize bacteriaespecially at high temperature and humidity. In addition, cell membranesare more permeable to external sugar alcohols than to external trehalose(Krallish I et al. 1997, Linders L J et al. 1997, Qiu L et al. 2000). Itis the synergetic effect of di-, and/or oligo-saccharides, such astrehalose, sucrose or inulin and sugar alcohols that provide betterprotection and improve cell viability over extended period of storage.Preferably, the concentration of the both saccharide and polyol in themixture is less than 60% w/v and more preferably less than 40% w/v ofthe preservation mixture. The ratio between the saccharide and thepolyol is preferably about 3:1, although a ratio of 1:3saccharide/polyol is also similarly effective in the preservation ofcertain probiotic species.

The present invention also provides methods of drying the mixture inglass form with a minimum loss of viability. It was discovered thatvitrifying and efficient drying of the preservation mixture under vacuumwas possible by foam formation as described by Bronshtein (2004). In thepresent invention, the need to foam the mixture is eliminated by forminga gel or cross-linking the polysaccharides in the preservation mixtureand slicing it to small pieces in order to dry it under vacuum. It alsoreduced the formation of a rubbery product which happened often in thefoaming process. Preferably, the preservation mixture, including theprobiotic, is allowed to gel at low temperature and is then sliced andvacuum dried under conditions suitable for glass formation. Morepreferably the polysaccharide in the mixture is selected from the groupof cross-linkable polysaccharides such as alginate, pectin or chitosan.The mixture is then extruded into Ca⁺⁺ bath and the strings or particlescollected, rinsed with water, and then soaked in a suitablesaccharide/polyol mixture followed by vacuum drying under conditionssuitable for glass formation.

The present invention also provides methods of vacuum drying thepreservation matrix without foaming or ice formation. The glassformation drying method comprises maintaining an initial vacuum of about2,500 mTOR for a period of time followed by drying at less than 100 mTORfor another period of time. The initial product temperature ispreferably maintained at or about −10 to about 40° C. during the periodat partially reduced pressure (2,500 mTOR) and then increased to about40 to 50° C., as the atmospheric pressure is decreased to less than 100mTOR. A final drying step at 20° C. under maximum vacuum (ca. 10 mTOR)for additional period of time can also be of benefit for the final waterremoval. The dry matrix can then be ground or milled and, if necessary,sieved to a desired particulate powder.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow diagram showing a method of counter-current desiccationof a wet matrix hydro gel using a powdered saccharide (trehalose) as astabilizing mixture.

FIG. 2 is a graph that depicts the effect of trehalose concentration inthe drying medium on bacteria viability. Maximal viability L. rhamnosuswas achieved at 0.5 M trehalose concentration. L. rhamnosus wasair-dried for 3 days in a laminar flow hood in the presence ofincreasing concentration trehalose.

FIG. 3 is a bar graph that depicts the effect of saccharides and polyols(at total concentration of 24% w/v in drying medium) on the after dryingviability of L. pracasei.

FIG. 4 is a bar graph that depicts the effect of different proportionsof saccharides/polyols (trehalose/mannitol or trehalose/isomalt) in amixture of polysaccharides (2% starch, 1% sodium alginate and 0.5%pectin) on viability of L. acidophilus after vacuum drying (the totalconcentration of the saccharides and polyols is 30% w/v).

FIG. 5 is a bar graph that depicts the effect of polysaccharide mix (2%starch, 1% sodium alginate and 0.5% pectin) with 3:1 trehalose/isomalt(the total concentration of the saccharides/polyols is 40% w/v) onviability of dry L. acidophilus in 45° C. at 0% or 33% relativehumidity.

FIG. 6 is a bar graph that depicts the effect of full stomach (12% nonfat skim milk, 2% glucose, 1% yeast extract and 0.05% cysteine; pH 2) orempty stomach (0.32% pepsin, 0.2% sodium chloride, pH 1.2) simulatedgastric juices on L. paracasei dried in free form or in glass form ofpolysaccharide/saccharide/polyol mix.

FIG. 7 is a bar graph that depicts the effect of empty stomach (0.32%pepsin, 0.2% sodium chloride, pH 1.2) simulated gastric juice on L.acidophilus dried in free form or in glass form ofpolysaccharide/saccharide/polyol mix.

FIG. 8 is a pair of graphs which depict the effect ofcarbohydrate/polysaccharide/egg albumen mix(trehalose/alginate/pectin/egg albumen 30:1.5:0.5:10) on viability ofdry Lactobacillus GG in 40° C. at 15% or 33% relative humidity.

FIG. 9 shows the effect of monosaccharide, disaccharide andoligosaccharides carbohydrates (trehlose, sucrose, galactose and inulin)on viability of dry Lactobacillus rhamnosus sp. in 33% relative humidityand at 30° C. or 40° C.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure relates to a composition that is a solid glass matrixcomprising a polysaccharide, saccharides, polyols and probiotic bacteriaand methods for the efficient large scale production of thiscomposition.

DEFINITIONS

As used herein, each of the following terms has the meaning associatedwith it in this section.

“Polysaccharides” refers to compounds consisting of a large number ofmonosaccharides linked with glycosidic bonds. As used herein, the termpolysaccharide refers only to those containing more than tenmonosaccharide residues.

“Saccharides” includes monosaccharides disaccharides andoligosaccharides.

“Polyols” refers in general to chemical compounds containing multiplehydroxyl groups. As used herein the term polyol means sugar alcohol,which is a hydrogenated form of carbohydrate, whose carbonyl group(aldehyde or ketone, reducing sugar) has been reduced to a primary orsecondary hydroxyl group. Some common sugar alcohols are: mannitol,sorbitol, xylitol, isomalt, maltitol, lactitol

“Vitrification” (i.e., glass formation) means formation of a glassy ornoncrystalline amorphous material. As used herein the term glass orglassy state means a liquid phase of such high viscosity and low watercontent that all chemical reactions are slowed to a near standstill, andthe bacteria cells become quiescent.

“Crystallization” refers to the formation of solid crystals from ahomogeneous solution. It is essentially a solid-liquid separation.

“Cryoprotectant” refers to a chemical or compound that is used toprevent the formation of ice crystals during the supercooling of a watercontaining mixture.

Fundamental to this invention is a polysaccharide capable of forming astrong gel matrix. This matrix preferably retains the bacteria and thepreservation mixture even after being sliced into small pieces or formedinto thin threads, strings, or particles. Additionally, thepolysaccharide matrix preferably possesses a controlled releasemechanism that protects the bacteria in the stomach, but is able torelease the bacteria at their site of action along the intestine.

Several polysaccharides exhibit these requirements and are suitable foruse as described herein. High amylose starch is a polysaccharide capableof forming firm gel after hydrating the starch granules in boilingwater, dispersing the granules with the aid of high shear mixer and thencooling the solution to about 0-10° C. The firmness and strength of thegel depend on the concentration of the starch in the solution, with amaximal workable concentration of up to 10% w/v. The sliced starch gelmatrix is also capable of retaining the live bacteria in thepreservation mixture, and since it is mostly non-digestible byintestinal or gastric juices, the bacteria are protected from gastricdestruction while within the starch matrix. The controlled releasemechanism is provided by the fact that high amylose starch is readilydigestible by the gut microflora at which time the delivered livebacteria are then released in their intact form.

Pectin is another suitable polysaccharide that performs very similar tohigh amylose starch. Pectin has an additional advantage since thestrength of the pectin gel matrix can be further increased by theaddition of divalent cations such as Ca⁺⁺ that forms bridges betweencarboxyl groups of the sugar polymers.

In a preferred embodiment of the present invention, alginate or acombination of alginate and non-digestible starch is used. Alginate canform a firm gel matrix by cross-linking with divalent cations. Thealginate containing preservation solution can be hardened into a firmgel matrix by internally cross-linking the alginate polysaccharides withCa⁺⁺ and then slicing the gel into small pieces while the bacteria andthe preservation mixture are fully retained within the gel matrix.Another method of cross linking the solution containing alginate andpreservation mixture is by extruding thin threads or strings of thesolution into Ca⁺⁺ bath. The strings harden instantly upon interactionwith Ca⁺⁺. The thin strings are harvested, rinsed with fresh water andthen soaked again in the preservation solution but without the presenceof polysaccharides. Another suitable method is to inject the thinthreads into Ca⁺⁺ bath, which also contains a preservation mixture atequal concentration and proportion of that of the extruded solution. Analternative method of preparation of the matrix is to spray atomize themixture into a bath containing Ca⁺⁺ cations. In such a procedure, smallmicroparticles from 50 to 500 microns are produced. Such particles areharvested, rinsed and soaked in the preservation medium, or the bathitself may contain the preservation mixture as described above for theproduction of thin threads or strings.

In one embodiment, the level of Ca⁺⁺ in the bath is constantly monitoredand only sufficient amount of cations necessary to cross link thealginate are added at a time. This eliminates the need to rinseexcessive Ca⁺⁺ from the strings or particles, thereby retaining all thesugar in the matrix, which would otherwise be washed away. In onepreferred mode of the present invention, monitoring the Ca⁺⁺ cationswithin a range of 0.25-0.5% w/v in the cross-linking bath is sufficientto harden the extruded alginate solution without any damage to theprobiotic bacteria. The gastric protection and controlled releasetrigger is also fulfilled by the use of alginate polysaccharide. Apolymeric matrix containing alginate remains firm in the acidicenvironment of the stomach, thereby protecting the bacteria, but quicklydisintegrates in the higher pH and phosphate-rich environment of theintestine. This results in the release of the probiotic bacteria attheir site of action along the intestine.

The purpose of the preservation mixture is to provide protection fromtemperature and moisture excursions of the final product without undueloss of viability of the probiotic bacteria. An ideal mixture contains acombination of saccharides and sugar alcohols that form an amorphousglassy phase with a glass transition temperature (Tg) well above ambienttemperature and water activity of the product. Trehalose alone is notalways sufficient to stabilize bacteria, especially at high temperatureand humidity. A more suitable mixture was found to be a combination oftrehalose and additional sugar alcohol that provides a synergetic effectof better protection and improved cell viability over extended periodsof storage. In addition to sugar alcohols and other long chainpolyalcohols, other preservation agents include sucrose, lacto sucrose,raffinose, maltodextrose, sepharose and dextran. These compounds maysynergistically improve the preservation of certain bacteria species.

The concentration and proportion of different carbohydrates in thepreservation mixture depends on several factors, but most particularlyon the bacteria species, strain, and drying conditions. The presentinvention discloses several optimal concentrations and sugar proportionssuitable for inclusion in the preservation mixture for a number ofprobiotic bacteria. Preferably, the carbohydrate concentration should beless than about 50%, as higher concentrations may interfere witheffective drying.

The preservation mixture optionally includes other additives thatcontribute to the overall stability of the probiotic bacteria. Suitableadditives include proteins, amino acids, diluents, chelating agents,buffers, preservatives, stabilizers, antioxidants, and lubricants.Specific examples of such additives would include, but are not limitedto: amino acids, lysine, glycine, L leucine, isoleucine, arginine,cysteine; proteins, human serum proteins, egg albumin, gelatin; buffers,various sodium phosphate buffers, citric/citrate buffers; preservatives,derivatives of hydroxybenzoic acids; antioxidants, vitamin E, ascorbicacid; lubricants, water miscible silicone/silicates; chelating agents,citric acid, EDTA, EGTA.

In a preferred mode of the present invention, the sliced gel or thinthreads or strings are dried in such a way that a glass is formed.Several drying methods can be employed, including, but not limited to,air drying at ambient temperature, spray drying, fluidized bed drying,vacuum drying, and freeze drying. As used herein, the dry glassypreservation mixture containing the dried bacteria cells preferablycontains a residual moisture content of less than about 5%, and, morepreferably, less than about 2%.

Drying is preferably performed under vacuum in a freeze drier at aproduct temperature above the freezing temperature of water under suchconditions. In general, vacuum drying are performed in two stages. Thefirst stage involves moderately reduced pressure (ca. 2500 mTOR), whilethe second stage involves lower pressures (i.e., higher vacuum −100mTOR) at higher product temperature (up to about 50° C.). This processcan be achieved using a programmable control system for vacuum pressureand shelf temperature. The vacuum and shelf temperature conditions forthe first drying stage are adjusted empirically according the size ofthe drier, heat transfer capacity, and the product load, but the goal isto keep the product above its freezing temperature while maximizing thewater evaporation rate. In one embodiment, the shelf temperature isinitially maintained at about 20° C. for about 16 hours, followed bygradually increasing the temperature to about 50° C. for the following48 hours. These drying conditions allow the formation of glassy statewherein the bacteria are locked in a quiescent state inside thepolysaccharide matrix.

In a preferred embodiment, the probiotic bacteria are dried as follows:the initial vacuum pressure is adjusted to about 2500 mTOR, with initialshelf temperature of 40° C. for 12 hours, followed by incrementallyreducing the atmospheric pressure (i.e., increasing the vacuum) to lessthan 100 mTOR at a rate of 125 mTOR/hr. Once the vacuum reaches 100mTOR, the sample is maintained at 40° C. for an additional 12 hours.Following this protocol, the drying procedure is completed within 48hours without substantially compromising viability. In accordance withthe present invention, the large surface area of the sliced and choppedgel or strings greatly increases evaporation rate without the need toboil or foam the product, thus eliminating inconsistent dryingconditions and splattering of the foaming product solution in the vacuumchamber. Additionally, the disclosed composition and method of dryingresults in a higher loading capacity of product as compared to the foamdrying method, that permits only a thin layer of solution to foam anddry efficiently.

An alternative drying procedure for the freshly prepared matrix stringsor particles includes a controlled desiccation of the matrix by additionof the hydrogel to a certain volume (preferably 1:10 by weight) of drypowdered saccharide such as trehalose or dry powdered preservationmixture. During this process, the hydrogel is rapidly desiccated atambient temperature, concentrating the preservation material in thematrix itself. The process is preferably set up in a counter-currentfashion where the fully hydrated hydrogel matrix containing the bacteriais added to one end of the process stream and fresh, dry powderedpreservation saccharide flows from the opposite direction (FIG. 1). Thewetted powdered saccharide material are dried at elevated temperatureand reused while the partially desiccated hydrogel then goes on to thesecond stage of vacuum drying described above. This processsignificantly reduces the drying time and process costs.

The resultant matrix-bound glass material containing the dried,stabilized probiotic bacteria has a Tg sufficiently high to preserve thebacteria at ambient temperature (up to 30° C.) in a relative humidity of33%. Generally, the higher the Tg, the higher the allowable storagetemperature and humidity. Tg of the dry glassy preservation mixture ofthe present invention is determined using standard techniques in theart, such as differential scanning calorimetry.

The methods and compositions of the invention facilitate the developmentof several products, including, but not limited to: live bacterialvaccines in a dry stable form, live bacterial nutraceuticals(probiotics) in a dry stable form, live bacterial starter cultures in adry stable form, live bacteria in a dry stable form for agricultural,aquaculture, or bioremedial use, and live bacterial cultures in a drystable form for the biotechnology industry.

The following examples illustrate various aspects of the presentinvention, relating to producing a delivery vehicle comprising a dry andstable matrix of polysaccharides, saccharides, polyols and probioticbacteria in a glass form. The compositions and drying methods areadapted to stabilize and preserve several probiotic bacteria in storageand gastric environment.

EXAMPLES

The subject matter of this disclosure is now described with reference tothe following Examples. These Examples are provided for the purpose ofillustration only, and the subject matter is not limited to theseExamples, but rather encompasses all variations which are evident as aresult of the teaching provided herein.

Example 1

High amylose starch (100 g Novation, National Starch and Chemical,Bridgewater, N.J.) was mixed with 150 ml of water at ambienttemperature. The starch mixture was then slowly added to 850 ml ofboiling water under vigorous mixing using a standard household blender.Once complete dispersion of the starch granules was observed (using abinocular microscope), the starch solution was allowed to cool and 300 gof trehalose and 100 g isomalt (both from Cargill Minneapolis, Minn.)were then dissolved in the mixture. Sodium alginate (15 g) was added tothe slurry and the entire mixture was allowed to cool to roomtemperature. Lactobacillus paracasei (200 g frozen paste direct fromfermentation harvest) was then mixed well into the slurry and the slurrywas extruded into a 1000 ml bath (held at 0-5° C.) containing 5 g CaCl₂,300 g trehalose and 100 g isomalt using a syringe equipped with 18 Gneedle. The CaCl₂ bath was gently stirred while injecting the slurry.The matrix strings were allowed to cross-link for 30 minutes and werethen harvested and blotted on paper towel. The composition of the gelmatrix is provided in Table 1.

TABLE 1 Gel matrix composition (g dry weight/100 g) a. High amylose (70%amylose) 10 g b. trehalose 30 g c. Isomalt 10 g d. Sodium Alginate 1.5 ge. L. paracasei 20 g f. Water 100 g

The thin threads were loaded on a tray (13×10 inch) and placed in afreeze drier (Virtis Advantage, Virtis, Gardiner, N.Y.). The condenserwas set to −70° C. and shelf temperature was set to 40° C. When theproduct had warmed up to the shelf temperature (measured by a pair oftemperature sensors plugged in the wet material), the vacuum wasinitiated and controlled at about 2500 mTOR with an external vacuumcontroller (Thyr-Cont, Electronic, GmbH). As the atmospheric pressuredecreased, the product temperature fell to and stabilized at about −2°C. After 12 hours, the product temperature had increased to about 10° C.At this point, the atmospheric pressure was dropped by about 500 mTORevery 4 hours until full vacuum pressure of 10 mTOR was established.Over this time period of increasing vacuum, the product temperature wascarefully maintained at or above −5° C. Twelve hours after establishingfull vacuum, the dried product was taken out of the freeze drier andground to fine powder using standard coffee grinder.

Example 2

100 g of trehalose and 300 g isomalt (both from Cargill Minneapolis,Minn.) were added to 1000 ml water and allowed to dissolve. Sodiumalginate (15 g) was mixed into the slurry and allowed to cool down toroom temperature. Lactobacillus paracasei (200 g frozen paste as inExample 1) was then added to the slurry, followed by 5 g of calciumphosphate dibasic and 5 g of gluconolactone. The slurry was allowed tocross-link at room temperature over the next 4 hours. The firm gel wassliced to thin and long threads through cheese grinder and blotted onpaper towel. The composition of the gel matrix is provided in Table 2.

TABLE 2 Gel matrix composition (g dry weight/100 g) a. trehalose 10 g b.Isomalt 30 g c. Sodium Alginate 1.5 g d. L. paracasei 20 g e. Water 100g

The thin threads were loaded on a tray (13×10 inch) and placed in afreeze drier for drying as outlined in example 1.

Example 3

300 g of trehalose (Cargill Minneapolis, Minn.) and 100 g mannitol(Sigma) were added to 1000 ml water and allowed to dissolve. Sodiumalginate (15 g) and pectin (5 g) were mixed into the slurry and theslurry was allowed to cool down to room temperature. Lactobacillusacidophilus (200 g frozen paste, directly from a fermentation harvest)was mixed well into the slurry. The slurry was then extruded through asyringe equipped with 18 G needle into 1000 ml bath (0-5° C.) containing5 g CaCl₂, 300 g trehalose and 100 g mannitol. The CaCl₂ bath was gentlystirred while extruding the slurry. The formed strings were allowed tocross-link for 30 minutes, harvested, and blotted on paper towel. Thecomposition of the gel matrix is provided in Table 3.

TABLE 3 Gel matrix composition (g dry weight/100 g) a. trehalose 30 g b.Mannitol 10 g c. Sodium Alginate 1.5 g d. Pectin 0.5 g e. L. acidophilus20 g f. Water 100 g

The thin threads were loaded on a tray (13×10 inch) and placed in afreeze drier for drying as outlined in example 1.

Example 4

Optimizing Trehalose Concentration in the Preservation Media

Dry powdered L. rhamnosus (LCS-742, Morinaga Milk Industry Co., LTD.,Kanagawa, Japan) was added to various concentrations of trehalose inbacterial culture media (L.MRS) and allowed to desiccate in a laminarflow hood at ambient temperature for 3 days. Bacteria viability as afunction of trehalose concentration was measured at the end of the 3-daydrying period. Dry bacterial powder or desiccated samples werereconstituted in sterile 50 mM PBS buffer pH 7.4. After homogenizing,solutions of reconstituted cultures were diluted (by 10-fold increments)in PBS buffer and plated in triplicate on L.MRS agar. After incubationat 35° C. for 48-72 hours, the number of colony forming units (CFU) wasdetermined and L. rhamnosus viability was found to be highest at aninitial trehalose concentration of 0.5 M (FIG. 2).

Example 5

The Effect of Different Sugar Alcohols on Drying Preservation of L.paracasei

L. paracasei was prepared and dried as described in example 2 exceptthat total sugar concentration was 24% and starch concentration was 2%in the preservation media. Several sugar alcohols were tested for theireffect on the bacteria viability after drying. FIG. 3 shows thattrehalose and sorbitol provided the best protection for the bacteriausing this drying and preservation process.

Example 6

The Effect of Different Sugar Proportions on Drying Preservation of L.acidophilus

L. acidophilus was dried as described in example 3 except that differentproportions of trehalose/mannitol or trehalose/isomalt were used and thefinal mixture contained a combination of 3 polysaccharides (2% starch,1% sodium alginate and 0.5% pectin). The viability of L. acidophilusafter vacuum drying is shown in FIG. 4. In all cases, the preservedbacteria had a far greater viability compared with bacteria driedwithout the saccharide/polyol mixtures, and the different ratios ofsaccharide to polyol used in the preservations mixtures yielded similarprotection capabilities for L. acidophilus.

Example 7

Stability of L. acidophilus in 45° C. at 0% or 33% Relative Humidity.

L. acidophilus was dried as described in example 6. The dried bacteriawas placed in temperature and humidity control incubator set at 45° C.and 0% relative humidity, or 45° C. and 33% relative humidity for 4hours. Viability of the bacteria was measured before and after thetemperature and humidity exposure. FIG. 5 shows that the polysaccharidemixture (2% starch, 1% sodium alginate and 0.5% pectin) providedadditional protection to that of trehalose/isomalt alone or freebacteria.

Example 8

Stability of the Composition of the Present Invention in SimulatedGastric Juices

L. acidophilus and L. paracasei were prepared and dried as described inexample 2. The dry powder matrix-glass bacteria was then exposed for 2hours to simulated gastric juice (full stomach—12% non fat skim milk, 2%glucose, 1% yeast extract and 0.05% cysteine; pH 2; or emptystomach—0.32% pepsin, 0.2% sodium chloride, pH 1.2). Bacterialviabilities were recorded before and after the exposure to the simulatedgastric juices. FIGS. 6 and 7 demonstrate a significant protection ofthe bacteria in the drying composition of the instant invention in thedifferent gastric conditions.

Example 9

300 g of trehalose (Cargill Minneapolis, Minn.) and 100 g egg albumen(Sigma) were added to 1000 ml water and allowed to dissolve. Sodiumalginate (15 g) and pectin (5 g) were mixed into the slurry and theslurry was allowed to cool down to room temperature. Lactobacillus GG(200 g frozen paste direct from fermentation harvest) was then added tothe slurry, followed by 5 g of calcium phosphate dibasic and 5 g ofgluconplactone. The slurry was allowed to cross-link at room temperatureover the next 4 hours. The firm gel was sliced to thin and long threadsthrough cheese grinder and blotted on paper towel. The composition ofthe gel matrix is provided in Table 4.

TABLE 4 Gel matrix composition (g dry weight/100 g) a. trehalose 30 g b.egg albumen 10 g c. Sodium Alginate 1.5 g d. Pectin 0.5 g e.Lactobacillus GG 20 g f. Water 100 g

The thin threads were loaded on a tray (13×10 inch) and placed in afreeze drier for drying as outlined in example 1.

Example 10

Stability of the Composition of the Present Invention in 40° C. and 15%or 33% Relative Humidity.

Lactobacillus GG was dried as described in example 9. The dried bacteriapowder was placed in temperature and humidity control incubator set at40° C. and 0% relative humidity, or 40° C. and 33% relative humidity for4 weeks. Viability of the bacteria was measured every 7 days. FIG. 8shows that the carbohydrates/polysaccharide/egg albumen mixture (30%trehalose, 10% egg albumen, 1.5% sodium alginate and 0.5% pectin)provided additional protection to that of trehalose/isomalt alone orfree bacteria.

Example 11

Preparation of Various Compositions Containing Monosaccharide,Disaccharide or Oligosaccharides Carbohydrates (Trehlose, Sucrose,Galactose or Inulin).

Four solutions containing galactose, sucrose, trehalose or inulin wereprepared. Each solution contained six (6) g of trehalose and 120 g ofisomalt (both from Cargill Minneapolis, Minn.) dissolved in 400 ml warmwater (40° C.). A saccharide (40 g of galactose, sucrose, trehalose orinulin) was mixed into the solution and the solution was allowed to cooldown to room temperature. Lactobacillus rhamnosus sp. (80 g frozen pastedirect from fermentation harvest) was then added to each solution,followed by 2 g of calcium carbonate and 2 g of gluconolactone. Theslurries were allowed to cross-link at room temperature over the next 4hours. The firm gels were sliced to thin and long threads through cheesegrinder.

The thin threads were loaded on trays and placed in a freeze drier(Model 25 SRC, Virtis, Gardiner, N.Y.). The condenser was set to −45°C., vacuum pressure applied at 2500 mTorr and shelf temperature adjustedto +40° C. As the atmospheric pressure decreased, the producttemperature fell to and stabilized at about −10 to around −1° C. Theprimary drying step was continued for 15 hours. The vacuum thenincreased by 300 mTORR every 2 h until full vacuum of about 100 mTORRestablished. A secondary drying step was then followed at full vacuumand shelf temperature maintained at 40° C. for additional 12 hours. Allcompositions were completely dried and water activity varied between0.223-0.253 Aw. The dried compositions were taken out of the freezedrier and ground to fine powder using standard coffee grinder.

Example 12

Viability of dry Lactobacillus rhamnosus sp. in 33% relative humidityand 30° C. or 40° C. Effect of compositions containing monosaccharide,disaccharide or oligosaccharides carbohydrates (trehlose, sucrose,galactose or inulin).

Lactobacillus rhamnosus sp. was dried as described in example 4. Thedried bacteria was placed in temperature and humidity control incubatorset at 30° C. or 40° C. and 33% relative humidity for 4 weeks and 5weeks, respectively. Viability of the bacteria was measuredperiodically. FIG. 9 show that the monosaccharide glactose didn'tprovide any protection as compared to a dry free bacteria compositioncontaining no sugar. The disaccharides trehalose and sucrose and theoligosaccharide inulin were equally effective and provided significantprotection of about 2-3 logs better viability after 28 days at 30° C.and 33% relative humidity or 35 days at 40° C. and 33% relativehumidity. Thus it is clear that sucrose is as effective as trehalose,and oligosaccharides are as effective as the disaccharide because theloss of viability of bacteria was low in all three examples.

REFERENCES

The following literature references are cited herein.

-   Bronshtein, V., C. Isaac, And G. Djordjevic. 2004. Preservation Of    Bacterial Cells At Ambient Temperatures, EP 1402003.-   Chen, T., J. P. Acker, A. Eroglu, S. Cheley, H. Bayley, A. Fowler,    and M. Toner. 2001. Beneficial effect of intracellular trehalose on    the membrane integrity of dried mammalian cells. Cryobiology 43:    168-81.-   Crowe, J. H., J. F. Carpenter, and L. M. Crowe. 1998. The role of    vitrification in anhydrobiosis. Annu Rev Physiol 60: 73-103.-   Crowe, L. M., and J. H. Crowe. 1992. Anhydrobiosis: a strategy for    survival. Adv Space Res 12: 239-47.-   Favaro-Trindade, C. S., and C. R. Grosso. 2002. Microencapsulation    of L. acidophilus (La-05) and B. lactis (Bb-12) and evaluation of    their survival at the pH values of the stomach and in bile. J    Microencapsul 19: 485-94.-   Franks, F., B. J. Aldous, and A. Auffret. 2003. Stable compositions,    U.S. Pat. No. 6,503,411.-   Isolauri E, Sutas Y, Kankaanpaa P, Arvilommi H, and S. S. 2001.    Probiotics: effects on immunity. Review. Am J Clin Nutr. 73:    444S-450S.-   Kailasapathy K, and C. J. 2000. Survival and therapeutic potential    of probiotic organisms with reference to Lactobacillus acidophilus    and Bifidobacterium spp. Review. Immunol Cell Biol. 78: 80-88.-   Krallish I, Jeppsson H₃ Rapoport A, and H.-H. B. 1997. Effect of    xylitol and trehalose on dry resistance of yeasts. Appl Microbiol    Biotechnol. 47: 447-51.-   Liao, Y. H., M. B. Brown, A. Quader, and G. P. Martin. 2002.    Protective mechanism of stabilizing excipients against dehydration    in the freeze-drying of proteins. Pharm Res 19: 1854-61.-   Linders L J₅ Wolkers W F, Hoekstra F A, and v. t. R. K. 1997. Affect    of added carbohydrates on membrane phase behavior and survival of    dried Lactobacillus plantarum. ECryobiology. 35: 31-40.-   Lievense L C, van 't Riet K. 1994. Convective drying of    bacteria. II. Factors influencing survival. Adv Biochem Eng    Biotechnol. 51:71-89.-   Marteau P R, de Vrese M, Cellier C J, and S. J. 2001. Protection    from gastrointestinal diseases with the use of probiotics. Review.    Am JCHn Nutr. 73: 430S-436S.-   Perdigon G, Fuller R, and R. R. 2001. Lactic acid bacteria and their    effect on the immune system. Review. Curr Issues Intest Microbiol.    2: 27-42.-   Qiu L, Lacey M J, and Bedding R A. 2000. Permeability of the    infective juveniles of Steinernema carpocapsae to glycerol during    osmotic dehydration and its effect on biochemical adaptation and    energy metabolism. Comp Biochem Physiol B Biochem MoI Biol. 125:    411-9.-   Roser, B. J., J. Kampinga, C. Colaco, and J. Blair. 2004. Solid dose    delivery vehicle and methods of making same, U.S. Pat. No.    6,811,792.-   Shah, N. P. 2000. Probiotic bacteria: selective enumeration and    survival in dairy foods. J Dairy Sd 83: 894-907.-   Simmons, D. L., P. Moslemy, G. D. Paquette, D. Guerin, and M.-H.    Joly. 2005. Stable probiotic microsphere compositions and their    methods of preparation, PA20050266069.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

While this subject matter has been disclosed with reference to specificembodiments, it is apparent that, other embodiments and variations canbe devised by others skilled in the. art without departing from the truespirit and scope of the subject matter described herein. The appendedclaims include all such embodiments and equivalent variations.

1. A dry composition in a solid glass form suitable for oral delivery,comprising a polysaccharide, one or more saccharides, a polyol, andprobiotic bacteria., wherein the ratio of saccharide to sugar alcohol is3:1 to 1:3 and wherein the sugar alcohol is selected from a groupconsisting of mannitol, glycerol, sorbitol, xylitol, maltitol, lactitoland isomalt.
 2. The composition, according to claim 1, wherein theprobiotic bacteria is selected from the group consisting of liveLactobacillus, Bifidobacterium, Enterococcus, Propionobacterium,Bacillus and Streptococcus.
 3. The composition, according to claim 1,wherein the polysaccharide is selected from starch, non-digestiblestarch, pectin, inulin, xanthan gum, alginate, alginic acid, chitosan,carrageenan, carboxymethyl cellulose, methyl cellulose, guar gum, gumarabic, locust bean gum and combinations thereof.
 4. The composition,according to claim 1, wherein the saccharides are selected from thegroup consisting of, disaccharides, trisaccharides and oligosaccharides.5. The composition according to claim 1 comprising a saccharide/polyolcombination is selected from the group consisting of trehalose/isomalt,sucrose/isomalt and inulin/isomalt.
 6. The composition according toclaim 1, further comprising suitable additives wherein the additive isselected from a group consisting of proteins, amino acids, buffers,preservatives and antioxidants.
 7. The composition, according to claim6, wherein the additive is selected from proteins and antioxidants andwherein the protein is albumen and the antioxidant is ascorbic acid. 8.The composition according to claim 6, further comprising apolysaccharide mixture comprising at least two polysaccharides selectedfrom starch, non-digestible starch, pectin, inulin, xanthan gum,alginate, alginic acid, chitosan, carrageenan, carboxymethyl cellulose,methyl cellulose, guar gum, gum arabic, and locust bean gum.
 9. Thecomposition according to claim 1, wherein the saccharide/polyol ratio isabout 3:1.
 10. The composition according to claim 1, wherein thesaccharide/polyol combination is selected from the group consisting oftrehalose/glycerol, trehalose/mannitol, trehalose/maltitol,trehalose/isomalt, trehalose/adonitol, trehalose/lactitol,trehalose/sorbitol, sucrose/glycerol, sucrose/mannitol,sucrose/maltitol, sucrose/isomalt, sucrose/adonitol, sucrose/lactitol,sucrose/sorbitol, inulin/glycerol, inulin/mannitol, inulin/maltitol,inulin/isomalt, inulin/adonitol, inulin/lactitol, and inulininulin/sorbitol.
 11. A process for producing a glass matrix, the processcomprising: a) dispersing under heating conditions at least onepolysaccharide in water; b) adding at least one saccharide, a sugaralcohol, an additive and a probiotic bacteria to form a slurry, whereinthe ratio of saccharide to sugar alcohol is 3:1 to 1:3 and wherein thesugar alcohol is selected from a group consisting of mannitol, glycerol,sorbitol, xylitol, maltitol, lactitol and isomalt; c) contacting theslurry with Ca⁺⁺ ions for a sufficient time to allow cross-linkingthereby forming a gel matrix, wherein the slurry further comprises asaccharide and sugar alcohol; d) slicing the gel matrix and placing samein a drier wherein the product temperature is maintained above thefreezing temperature; e) reducing the pressure during a first dryingstage and maintaining the product temperature from about (−) 10 to about(+) 40° C. for a first period of time; and f) further reducing thepressure during a second drying stage and increasing the producttemperature to between about (+) 40 to about (+) 50° C. for a secondperiod of time.
 12. The process according to claim 11, wherein thepressure during the first stage is reduced to about 2,500 mTOR and thepressure at the second stage is maintained less that 100 mTOR.
 13. Theprocess according to claim 11, wherein the first period of time is from12 to 16 hours and the second period time is from 12 to 48 hours. 14.The process according to claim 11, wherein contacting the slurry of stepc) comprises extruding the slurry into the Ca++ ion containing bath andforming matrix strings that crosslink while retained in the bath. 15.The process according to claim 11, wherein the gel matrix of step c) isformed by internal cross linking of the polysaccharide with C++ ions andslicing the gel to form threads before placement in the drier.
 16. Theprocess according to claim 11, wherein the probiotic bacteria isselected from the group consisting of live Lactobacillus,Bifidobacterium, Enterococcus, Propionobacterium, Bacillus andStreptococcus.
 17. The process according to claim 11, wherein thepolysaccharide is selected from starch, a non-digestible starch, pectin,inulin, xanthan gum, alginate, alginic acid, chitosan, carrageenan,carboxymethyl cellulose, methyl cellulose, guar gum, gum arabic, locustbean gum and combinations thereof.
 18. The process according to claim11, wherein the saccharides are selected from the group consisting ofdisaccharides, trisaccharides and oligosaccharides.
 19. The processaccording to claim 11, wherein the saccharide/polyol combination isselected from the group consisting of a saccharide/polyol combinationselected from the group consisting of trehalose/isomalt, sucrose/isomaltand inulin/isomalt.
 20. The process according to claim 11, wherein thesaccharide/polyol ratio is about 3:1.